Molecular Biochemistry II

Amino Acid Catabolism: Nitrogen

Transaminases
(aminotransferases) catalyze the reversible reaction shown at right. There
are multiple transaminase enzymes which vary in
substrate specificity. Some show preference for particular amino acids or classes of amino acids as
amino group donors and/or for particular a-keto acid acceptors.

In another example shown at right, alanine becomes pyruvate as the amino
group is transferred to a-ketoglutarate.

Transaminases equilibrate amino groups
among available
a-keto acids. This permits synthesis of non-essential
amino acids, using amino groups derived from other amino acids and carbon
skeletons synthesized in the cell. Thus a balance of different amino acids is
maintained, as proteins of varied amino acid contents are synthesized.

Although the amino N of one amino acid can be used to synthesize another amino acid, nitrogen must be obtained in the diet as amino acids
(proteins).

Essential amino acids must be consumed in the diet
because mammalian cells lack the enzymes to synthesize their carbon skeletons (a-keto acids). These include:

Isoleucine, leucine, & valine

Lysine

Threonine

Tryptophan

Phenylalanine (Tyrosine can be made from phenylalanine.)

Methionine (Cysteine can be made from methionine.)

Histidine (Essential for infants.)

The prosthetic group of the transaminase
enzyme is pyridoxal phosphate (PLP), a derivative of vitamin B6.
(p. 986).

In the "resting" state, the aldehyde group of pyridoxal phosphate is in a Schiff base linkage to the e-amino
group of an enzymelysine
side-chain.

The a-amino group of a substrate
amino acid displaces the enzyme lysine, to form a Schiff base linkage to
PLP.

The active site lysine extracts a proton,
promoting tautomerization (shift of the double bond), followed by reprotonation with hydrolysis.

What was an amino
acid leaves as an a-keto
acid. The amino group remains on what is now pyridoxamine
Phosphate (PMP).

A different a-keto acid reacts with PMP, and the process
reverses, to
complete the reaction.

For more details about the reaction
mechanism, and the postulated role of molecular strain in catalysis, see the
article by Hayashi et al.

Several other enzymes that catalyze metabolism or synthesis of amino acids
also utilize PLP as prosthetic
group, and have mechanisms involving a Schiff base linkage of the amino group to PLP.

Chime Exercise: Two
neighboring students or student groups should team up, each displaying, as recommended, one of the following:

Transaminase with PLP in Schiff base
linkage to the active site lysine residue.

Transaminase in the PMP form, with
glutarate, an analog of a-ketoglutarate, at the active site.

Students should then show and explain the structure displayed by
them to the neighboring student or student group.

Transaminase-PLP

Transaminase-PMP

In addition to equilibrating amino groups among available
a-keto acids, transaminases funnel amino groups
from excess dietary amino acids to those amino acids (e.g., glutamate) that can
be deaminated. Carbon skeletons of
deaminated amino acids can be catabolized for energy or used to synthesize
glucose or fatty acids for energy storage.

Only a few amino acids can be deaminated directly. Glutamate Dehydrogenase
catalyzes a major reaction that effects net removal of N from the amino
acid pool .

Glutamate Dehydrogenase is one of the few enzymes that
can utilize either NAD+ or NADP+ as electron
acceptor.

Oxidation at the a-carbon is followed by
hydrolysis, releasing NH4+. (See diagram at right and on p.
989.)

At right is summarized the role of transaminases in funneling amino N
to glutamate, which is deaminated via Glutamate Dehydrogenase, producing NH4+.

The reaction, which involves cleavage of 2 ~P bonds of ATP, is essentially irreversible.

Ammonia is the nitrogen input for this reaction of the Urea
Cycle.

Alternate forms of Carbamoyl Phosphate Synthase, designated Types II
and III, initially generate ammonia by hydrolysis of glutamine. The type II enzyme includes a long internal tunnel through which
ammonia and reaction intermediates such as carbamate pass from one active
site to another.

Carbamoyl Phosphate Synthase is the
committed step of the Urea Cycle, and is subject to regulation.

Carbamoyl Phosphate Synthase has an absolute requirement for an allosteric
activator
N-acetylglutamate (p. 995). This derivative of glutamate is
synthesized from acetyl-CoA and glutamate when cellular [glutamate] is high, signaling an
excess of
free amino acids due to protein breakdown or dietary intake.

Oxaloacetate is converted to aspartate via transamination,
e.g., from glutamate (see above). Aspartate then reenters the Urea Cycle, carrying an amino
group derived from another amino acid.

Hereditary deficiency of any of the Urea Cycle
enzymes leads to hyperammonemia - elevated [ammonia]
in blood. Total lack of any Urea Cycle enzyme is lethal. Elevated ammonia is very
toxic, especially to the brain. If not treated immediately after birth, severe mental
retardation results.

Postulated mechanisms for toxicity of high [ammonia]:

High [ammonia] would drive the
Glutamine
Synthase reaction (p. 1033).glutamate + ATP + NH3àglutamine + ADP + PiThis would deplete glutamate, a neurotransmitter and the precursor for synthesis of
GABA, another neurotransmitter.

Depletion of glutamate, as well as the high ammonia level, would drive
the Glutamate Dehydrogenase reaction to reverse.glutamate + NAD(P)+ßa-ketoglutarate +
NAD(P)H + NH4+The resulting depletion of a-ketoglutarate, an essential
Krebs Cycle intermediate, could impair energy metabolism in the brain.

Treatment of deficiency of Urea Cycle
enzymes (some treatments depend on which enzyme is deficient):

limiting protein intake to the amount
barely adequate to supply amino acids for growth, while adding to the diet the a-keto acid analogs of essential amino acids.

Liver transplantation has also been
used, since liver is the organ that carries out Urea Cycle.

Explore information about such genetic diseases in the OMIMweb site (Online Mendelian Inheritance in Man).
Links are provide here to
information relating to hereditary
deficiencies of:

Use the menu at the top of the OMIM page to change the
display to Clinical Synopsis or Detailed. Within the Detailed
display, you may choose to view listed items such as Clinical Features, and
Biochemical Features.

Other roles of Urea Cycle intermediates:

The complete Urea Cycle occurs significantly
only in liver. However some
enzymes of this pathway are expressed in other cells and tissues, where they
function to generate arginine and
ornithine, which are precursors for other
important molecules. For example, Argininosuccinate
Synthase (see above), which catalyzes synthesis of the precursor to arginine, is found in
most tissues. A mitochondrial enzyme Arginase II,
distinct from the cytosolic Arginase involved in the Urea Cycle, cleaves
arginine to yield ornithine.

The amino acid arginine, in addition to being a constituent of
proteins and an intermediate of the Urea Cycle, serves as precursor for synthesis of
creatine
and the signal molecule nitric oxide.

Synthesis of the radical species nitric oxide (·NO) from
arginine is
catalyzed Nitric Oxide Synthase, a
distant relative of cytochrome P450.
Different isoforms of Nitric Oxide Synthase (e.g., eNOS expressed in
endothelial cells and nNOS in neuronal cells) are subject to differing
regulation.

Nitric oxide (·NO) is a short-lived signalmolecule with diverse roles in different
cell types, including regulation of smooth muscle contraction, gene
transcription, metabolism, and neurotransmission. Many of the regulatory effects of ·NO
arise from its activation of a soluble cytosolic Guanylate Cyclase enzyme that
catalyzes synthesis of cyclic-GMP
(analogous in structure to cyclic-AMP).

Cytotoxic effects of ·NO observed under
some conditions are attributed to its non-enzymatic reaction with superoxide (O2·-)
to form the strong oxidant peroxynitrite. (ONOO-).

Polyamines
include putrescine, spermidine, and spermine.
Ornithine is a major precursor
for synthesis of polyamines. Conversion of ornithine to putrescine is
catalyzed by Ornithine Decarboxylase.

There is no tRNA for citrulline,
and this amino acid is not incorporated translationally into proteins.
However, Ca++-activated
Peptidylarginine Deiminases convert
arginine residues within proteins to citrulline
as a post-translational modification.

The substitution of citrulline,
which lacks arginine's positive charge, may alter structure
and properties such as binding affinities of a protein. For
example, citrullination of certain proteins, including keratin
intermediate filament proteins, is essential to terminal differentiation of skin cells.

Excessive protein citrullination, with production of antibodies against citrullinated proteins,
is found to be a factor in the autoimmune
diseases such as rheumatoid arthritis and multiple sclerosis.